Thin layer chromatography

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Thin Layer Chromatography

Thin Layer Chromatographylavakusa Naik BanavatuAnalytical Chemistry, Andhra UniversityChromatographyChromatographyis a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.Types of Chromatographic Techniques:TechniqueStationaryMobile PhaseColumn/Adsorption ChromatographysolidLiquidPartition ChromatographyLiquidLiquidPaper ChromatographyLiquidLiquidThin Layer Chromatography (TLC)Liquid/SolidLiquidGas Liquid chromatography (GLC)LiquidgasGas Solid Chromatography (GSC)Solid gasIon Exchange ChromatographySolidLiquidIntroductionTLC is one of the simplest, fastest, easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds.

TLC is a form of liquid chromatography consisting of:A mobile phase (developing solvent) andA stationary phase (a plate or strip coated with a form of silica gel)Analysis is performed on a flat surface under atmospheric pressure and room temperatureDefinitionsThin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a glass plate and liquid as a mobile phase.Synonyms: Drop, strip, spread layer, surface chromatography and open column chromatography - Adsorption or retention or partition or both or any other principle of a substance (s ) on the stationary phase- Separation of the adsorbed substances by the mobile phase- Recovery of the separated substances by a continuous flow of the mobile phase (elution)- Qualitative and quantitative analysis of the eluted substances

HistoryMichael Tswett is credited as being the father of liquid chromatography. Tswett developed his ideas in the early 1900s.

1938:- Izmailov & shraiber described basic principle and used it for separation of plant extracts.

1944:- Consden, Gorden & Martin started using filter papers for separation of amino acid.

1950:- Kirchner who used impregnated glass plate coated with alumina, identified terpenes.

1958:- Ergon stahl introduced a standard equipment for preparing uniform thin layers of known thicknessWhen TLC used ?

Where TLC used

Principle of TLCIt is based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed

The components with more affinity towards stationary phase travels slower.

Components with less affinity towards stationary phase travels fasterIn TLC, a solid phase, the adsorbent, is coated onto a solid support (thin sheet of glass, plastic, and aluminum ) as a thin layer (about 0.25 mm thick). In many cases, a small amount of a binder such as plaster of Paris is mixed with the absorbent to facilitate the coating. The mixture (A + B) to be separated is dissolved in a solvent and the resulting solution is spotted onto the thin layer plate near the bottom. A solvent, or mixture of solvents, called the eluatant, is allowed to flow up the plate by capillary action. At all times, the solid will adsorb a certain fraction of each component of the mixture and the remainder will be in solution. Any one molecule will spend part of the time sitting still on the adsorbent with the remainder moving up the plate with the solvent. A substance that is strongly adsorbed (say, A) will have a greater fraction of its molecules adsorbed at any one time, and thus any one molecule of A will spend more time sitting still and less time moving and vice versa.

Continue.Separation of mixtures in microgram quantities by movement of a solvent across a flat surface; components migrate at different rates due to differences in solubility, adsorption, size or charge; elution is halted when or before the solvent front reaches the opposite side of the surface and the components examined in situ or removed for further analysis.Continue..Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase

1.The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.

2.The mobile phase: is a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to: how much they adsorb on the stationary phase versus how much they dissolve in the mobile phaseContinue.....Basic Theory


ContinueFactors affecting Rf valueIt depends on following factors:Nature adsorbentMobile phaseActivityThickness of layerTemperatureEquilibriumLoadingDipping zoneChromatographic techniques Selection of Stationary PhaseThe choice of the stationary phase for a given separation problem is the most difficult decision in TLCThe chose of stationary Phase in following characters considered.

The chemical composition of the stationary Phase and in particular that of its surface, must be suitable for the task. To obtain satisfactory separation efficiency, the mean particle size, the particle size distribution and the morphology of the particle must be consideredStationary phases for thin-layer chromatography

Mobile PhaseThe choice of mobile phase is largely empirical but general rules can be formulated. A mixture of an organic solvent and water with the addition of acid, base or complexing agent to optimize the solubility of the components of a mixture can be used. For example, good separations of polar or ionic solutes can be achieved with a mixture of water and n-butanol. Addition of acetic acid to the mixture allows more water to be incorporated and increases the solubility of basic materials, whilst the addition of ammonia increases the solubility of acidic materials. If the stationary phase is hydrophobic, various mixtures of benzene, cyclohexane and chloroform provide satisfactory mobile phases. It should be emphasized that a large degree of trial and error is involved in their selection. For TLC on silica gel, a mobile phase with as low a polarity as possible should be used consistent with achieving a satisfactory separation. Polar solvents can themselves become strongly adsorbed thereby producing a partition system, a situation which may not be as desirableMobile PhaseThe eluting solvent should also show a maximum of selectivity in its ability to dissolve or desorbs the substances being separated.A more important property of the solvent is its ability to be itself adsorbed on the adsorbent.A number of common solvents in approximate order of increasing adsorb ability, and hence in order of increasing eluting power.Mixtures of solvents can be used and, since increasing eluting power results (0.5 to 2% by volume)solvents to be used in chromatography should be quite dryLeast Eluting Power (alumina as adsorbent) -Petroleum ether (hexane; pentane)-Cyclohexane-Carbon tetrachloride-Benzene-Dichloromethane-Chloroform ; -Ether -Ethyl acetate (anhydrous)-Acetone (anhydrous)-Ethanol ; -Methanol-Water ;-PyridineGreatest Eluting Power (alumina as adsorbent)- Organic acidsSelection of adsorbentsSolubility of compound e.g, hydrophilic or lipophilicNature of substance to be seperated i.e whether it is acidic, basic or amphotericAdsorbent particle sizeAdsorbent should not adhere to glass plateReactivity of compound with the solvent or adsorbentChemical reactivity of compounds with binders

That in experiments performed to solve various problems by the adsorptionmethod the use of various sorbents would be necessary. They testedvarious substances, including aluminum oxides, aluminum silicates, calcium carbonate,kaolin, kieselguhr, magnesium oxide, powdered sugar, silica gels, starch and talcThe separation efficiency obtained in TLC is essentially determined by the mean particle size and the size distribution of the sorption agent used in the preparation of the layer. As can be seen from Fig. Below, the mean particle size of silica gel of a quality suitable for HPTLC is 5 m, that of TLC quality ca. 11 m and that of PSC quality over 20 m.Chromatographic media-coating material

What Are the Uses of Precoated LayersTLC investigations are mainly concerned wit the determination of

IdentityPurityAssayor a combination of these parametersPREPARATION OF CHROMATOPLATESGlass plates or flexible plates are commonly used for adsorbent. Size used depends on type of separation to be carried out, the type of chromatographic tank and spreading apparatus available.The standard sizes are 20 x 5 cm, 20 x 10 cm or 20 x 20 cm .The surface should be flat without irregularities. The standard film thickness is 250um

8 January 2015Dept. of Pharmaceutics33Methods for application of adsorbent.PouringDippingSprayingSpreading.8 January 2015Dept. of Pharmaceutics34Pouring: The adsorbent of finely divided and homogeneous particle size is made into slurry and is poured on a plate and allowed to flow over it so that it is evenly covered.

Dipping : This technique is used for small plates by dipping the two plates at a time, back to back in a slurry of adsorbent in chloroform or other volatile solvents. Exact thickness of layer is not known and evenness of layer may not be good.8 January 2015Dept. of Pharmaceutics35Spraying : Slurry is diluted further for the operation of sprayer. But this technique is not used now a days as it is difficult to get uniform layer.

Spreading : All the above methods fail to give thin and uniform layers. Modern methods utilize the spreading devices for preparation of uniform thin layers on glass plates. Commercial spreaders a